The lack of specific cellular markers for quantitative assessment of pathogenic damage to the blood-brain barrier (BBB), which is mainly constituted by microvascular endothelial cells (BMEC), is one of the most challenging issues in central nervous system (CNS) disorders induced by various pathogenic insults, including AIDS, and the use of illegal drugs [e.g., methamphetamine (METH), marijuana, opiates] and legal substances such as alcohol and nicotine (NT), a major component of environmental tobacco smoke. The incidence of NeuroAIDS is higher or accelerated among drug users and tobacco smokers. Alterations of brain microvasculature and the disruption of the BBB integrity are commonly associated with the use of drugs and HIV-1 infection in the era of HAART. In our previous work it was found that dysfunction of BMEC could be induced by NT, meningitic pathogens and microbial factors, including HIV-1 virulence factors (VFs) gp41 and gp120. The overall goal of this pilot project is to perform feasibility studies on the development of new cell-based biomarkers of the BBB disorders induced by drug abuse (NT) and HIV VFs (gp120 and gp41) through a combination of in vitro(BMEC)/in vivo (mouse) BBB models and high throughput single-cell profiling approaches. Our studies have shown that mice treated with NT and gp120 resulted in a synergistic increase in blood levels of circulating BMEC (cBMEC) [UEA+(EC marker)/CD146+(EC marker)/S100B+ (brain marker)] as well as endothelial progenitor cells (EPC)[UEA+/CD146+/CD133+(progenitor cell marker)]. NT and gp120 were able to significantly increase the serum levels of UCHL1 (a new BBB marker) as well as S100B in mice, which are correlated with the changes in cBMEC and EPC. Based on these findings, we have hypothesized that cBMEC in the peripheral blood, which are endowed with a full-blown BBB phenotype, are dynamically shed from the BBB. The replacement of shed or dead BMEC could occur through division of surrounding EC and homing of EPC, which are derived from bone marrow. The integrity of the BBB is associated with the functional changes in the brain microvasculatures and maintained by the balance between BMEC shedding/death rate and cell renewal rate. Peripheral blood cBMEC detected by molecular and single-cell profiling approaches can be used as a novel genomewide, cell-based multiple biomarker and gene network detection tool for the BBB injury, which is induced by NT, gp120 and other pathogenic insults. The availability of cBMEC as peripheral blood biomarkers in the CNS diseases will have a great impact in neuroscience research on drug abuse and neuroAIDS. Our hypotheses will be tested with the preclinical proof-of-concept studies through the following two Specific Aims: (1). Establish in vitro/in vivo experimental approaches for the characterization of cBMEC and UCHL1 as novel biomarkers of BBB injury induced by NT and HIV-1 gp120. (2). Determine the correlation between disease-specific BBB injury and blood levels of biomarkers by molecular typing and single-cell profiling of cBMEC.